Bioactive-Guided Phytochemical Investigations, In Vitro and In Silico Alpha-Glucosidase Inhibition of Two Vietnamese Medicinal Plants Dicranopteris linearis and Psychotria adenophylla

Little is known about the chemical and biological profiles of Dicranopteris linearis and Psychotria adenophylla. No previous studies have investigated alpha-glucosidase inhibition using extracts from D. linearis and P. adenophylla. In this paper, bioactive-guided isolation procedures were applied to the plants D. linearis and P. adenophylla based on alpha-glucosidase inhibition. From the most active fractions, 20 compounds (DL1–DL13 and PA1–PA7) were isolated. The chemical structures were elucidated using spectroscopic data and compared with those available in the literature. These compounds were evaluated for alpha-glucosidase inhibition, while a molecular docking study was performed to elucidate the mechanisms involved. Consequently, D. linearis and P. adenophylla might serve as a good potential for developing new antidiabetic preparations.

The genus Psychotria (Rubiaceae) comprises approximately 1700 species, popularly distributed in tropical and subtropical areas [8].Different parts of these species (leaves, roots, and rhizomes) have been traditionally used to treat fever, bronchitis, ulcers, stomachaches, and gynecological hemorrhage in females [9].Pharmacological studies have indicated that Psychotria plants exhibit various biological activities, including antimicrobial, antiviral, analgesic, hypoglycemic, and strong cytotoxic activities against several cancer cell lines [10,11].The chemical data of the genus Psychotria have been comprehensively studied, indicating that the major compounds of this genus are alkaloids and terpenoids.Some alkaloids are the biomarkers of Psychotria plants [12].Psychotria adenophylla wall is distributed in the south of Vietnam, and the phytochemical data on this plant are scarce.There has been only one report about the chemical constituents of P. adenophylla growing in India, which indicated the presence of eight sterols and triterpenes, including β-sitosterol, betulin, betulinic acid, α-amyrin, ursolic acid, friedelin, bauerenol, and bauerenol acetate [13].
There is limited information available concerning the chemical and biological profiles of D. linearis and P. adenophylla.Additionally, there have been no prior investigations into the potential of extracts from these plants to inhibit alpha-glucosidase.During our systematic research on alpha-glucosidase inhibitors from Vietnamese medicinal plants, bioactiveguided isolation procedures were applied to the plants D. linearis and P. adenophylla based on alpha-glucosidase inhibition.Twenty compounds (DL1-DL13 and PA1-PA7) were isolated from the most active fraction (Figures 1 and 2).The chemical structures were elucidated using spectroscopic data and compared with those available in the literature.These compounds were evaluated for alpha-glucosidase inhibition, and a molecular docking study was performed to elucidate the mechanisms involved.

Results
Extracts/fractions from D. linearis and P. adenophylla were evaluated for alpha-glucosidase inhibition (Table 1).The most bioactive extract of each plant was selected for further isolation.

Alpha-Glucosidase Inhibition of Extracts, Fractions, and Compounds from D. linearis and P. adenophylla
The results of alpha-glucosidase inhibition for both extracts and fractions obtained from D. linearis and P. adenophylla are presented in Table 1.Additionally, the evaluation of the isolated compounds for their alpha-glucosidase inhibitory activity is presented in Table 2  The molecular docking models of DL1 [34], DL2 [35], DL6 [36], DL10 [37], DL11 [38], DL12 [39], DL13 [39], and PA4 [40] were investigated extensively, so no in silico analysis of those compounds were performed in the current study.Molecular docking studies were applied to compounds DL3, DL5, DL8, PA5, and PA6.The results are shown in Table 3 and Figures 4 and 5.

Chemical Composition of D. linearis and P. adenophylla
Until now, 21 compounds have been reported previously for D. linearis (see Figure S21).Most of them are glycosides with 1-3 sugar units, and they appear in polar fractions.From the Vietnamese D. linearis, three steroids (DL1-DL3), eight flavonoids (DL6-DL13),

Chemical Composition of D. linearis and P. adenophylla
Until now, 21 compounds have been reported previously for D. linearis (see Figure S21).Most of them are glycosides with 1-3 sugar units, and they appear in polar fractions.From the Vietnamese D. linearis, three steroids (DL1-DL3), eight flavonoids (DL6-DL13), one diphenylketone (DL4), and an 8-member-ring compound (DL5) were isolated.In 2021, Zakaria and co-workers undertook multiple analytic methods to determine phenolic compounds and triterpenes from the Malaysian Dicranopteris linearis leaves without isolation [41].To the best of our knowledge, compounds DL1 and DL3-DL10 were reported for the first time in the genus Dicranopteris.
Only six compounds were reported previously for P. adenophylla (see Figure S22).All of them are common compounds found in many plants.Using bioactive-guided isolation on the Vietnamese P. adenophylla, eight compounds were successfully isolated.To the best of our knowledge, compounds PA1-PA6 were reported for the first time in the genus Psychotria.Many previous phytochemical investigations on the Psychotria plants focused on alkaloids using an alkaloid-isolation procedure [42][43][44][45].Interestingly, the alkaloid tribulusimide D (PA6) was detected in P. adenophylla, representing a new alkaloid type within the genus Psychotria.

Alpha-Glucosidase Inhibition of Extracts, Fractions, and Compounds from D. linearis and P. adenophylla
Our literature review showed that crude extracts of both D. linearis and P. adenophylla have not been evaluated for alpha-glucosidase inhibition.Very little is known about the alpha-glucosidase inhibitory activity of the Dicranopteris and Psychotria plants.Recent studies regarding the alpha-glucosidase inhibition of the crude extracts from other Dicranopteris and Psychotria plants, D. caudata, P. malayana, and P viridiflora, were performed, indicating the potent inhibition of the extracts of these plants [46][47][48].
Although the crude methanol extract of D. linearis showed good activity, its derived extracts and fractions showed weaker alpha-glucosidase inhibition (Table 1).This indicated that the combination of all components of D. linearis might increase the activity.Further antidiabetic investigation of this plant should be conducted on the crude extract.
As seen in Table 1, the ethyl acetate extract of D. linearis had an IC 50 value of 124.1 µg/mL.Therefore, it was further fractionated to obtain five fractions (EA1-EA5).Fractions EA2-EA4 were chosen for further isolation based on their good alpha-glucosidase inhibition.Compounds DL7 and DL10-DL13 were isolated from these fractions.Two isoflavones, DL6 and DL7, were isolated from the extract HEA.Those mentioned flavonoids are wellknown alpha-glucosidase inhibitors that have been comprehensively studied.Particularly, the consistency between in vitro alpha-glucosidase inhibitory activity and in vivo data of luteolin (DL10) and daizein (DL6) was confirmed by various reports [37,49,50].Quercetin (DL11), isoquercitin (DL12), and kaempferol (DL13) were potent inhibitors, and the two formers were non-competitive types [39,51].Two isoflavones, genistein (DL5) and daizein (DL6), also inhibited alpha-glucosidase, which is consistent with previously published reports [36,52].These compounds are abundant in the ethyl acetate extract of D. linearis, indicating that they determine the activity of the extract EA.Multi-oxygenated flavonoids DL8 and DL9 are less potent than the above-mentioned flavonoids.Their low inhibition might be affected by the presence of the 3-oMe group, which was reported previously by Nguyen et al., 2023 [53].
The EA extract and its derived fractions of P. adenophylla showed potent activities, with IC 50 values ranging from 1.7 to 26.6 µg/mL, much lower than that of the crude MeOH extract (Table 1).However, the isolation of bioactive components from these extracts and fractions is limited due to their high lignin content.The detection of the mixture of undefined lignins was determined using NMR and HPLC methods (Figure S20.1-20.3).Such macromolecules were known to be potent alpha-glucosidase inhibitors [54].Only three compounds, PA5-PA7, were isolated from the most active fraction, EA5, but these compounds do not reflect the activity of the starting fraction.These compounds showed moderate activity with IC 50 values in the range of 76.2-249.7 µM.Tribulusimide D (PA6) was previously found in Euphorbia dracunculoides [55] and Tribuli fructus [32], showing significant hepatoprotective activity with an EC 50 value of 13.46 µM.
The docking results showed that 5/7 ligands were able to bind to residues within the binding site of alpha-glucosidase.The steric effect with bulky groups prevented complexation with proteins of 2,2 ,4,4 -tetra-tert-butyl benzophenone and 6-hydroxy stigmast-4-en-3-one.In order of docking score, acarbose had the lowest value of −17.3 kcal/mol while the studied compounds ranged from −7.4 to −4.3 kcal/mol.In terms of energy estimates using MM-GBSA, acarbose showed the lowest value at −128.2 kcal/mol, followed by 6-hydroxy stigmast-4-en-3-one (DL3) at -65.4 kcal/mol, and tribulisimide D (PA6) and jaceidin (DL8) in the same range at −43 kcal/mol.Chakyuglupulin B (DL4) exhibited a higher value of −29.3 kcal/mol.Chrysophanol (PA5) had the highest energy estimate at −24 kcal/mol.These results show some deviation from the experimental values, but overall, the experimental values of all the studied compounds fall within the range of −5.5 to −4.5 kcal/mol.This indicates that these compounds exhibit moderate inhibition of Saccharomyces cerevisiae alpha-glucosidase.
In terms of interactions, acarbose stood out due to its extensive occupancy of the chemical space and the significant presence of hydrogen bonding within the interaction networks.The inhibitory mechanism of acarbose ignited with the H-bond between the hydroxyl group of acarbose and Asp 214, whereas Glu 276, the residue responsible for catalyzing the hydrolysis of the normal 1,4-alpha-gluco bond, was engaged with two hydrogen bonds, one from acarbose's nitrogen and one from the hydroxyl group.In addition, a salt bridge was also formed between the cationic ammonium and the carboxylate group of Glu 276.This observation was only visible when the nitrogen of acarbose was protonated in a physiological environment (pH = 7 ± 2), which was achieved using Ligprep.Therefore, Glu 276 could not hydrolyze the C-N bond between the glucose molecules of acarbose.Additionally, Asp 349, a residue considered a transition state stabilizer of diose or triose degradation, could also form a hydrogen bond with acarbose.According to the described mechanism, Figures 4 and 5 and Table 3 indicate that among the three key residues (Asp 214, Glu 276, and Asp 349), all the studied compounds formed hydrogen bonds with either Asp 214 or both Asp 214 and Glu 276, except for compound B, which only formed a H-bond with the key residue Asp 349.This provides compelling evidence of their potential inhibitory effect on the enzyme's activity, potentially impeding polysaccharide hydrolysis by Saccharomyces cerevisiae alpha-glucosidase.
The moderate activity of these ligands may be attributed to the specific structure of each substance, which prevents them from optimally filling the chemical space within the alpha-glucosidase binding site.As a result, there are easily accessible solvent regions deep inside the binding site, which reduces their inhibitory activity.

Source of the Plant Material
D. linearis leaves were collected in Ba Ria-Vung Tau Province, Vietnam, from June to July 2022.The scientific name was identified as Dicranopteris linearis (Burm.F.) Underw.by Dr. Dang Van Son (deposited as No UE-P017).
The leaves of P. adenophylla were collected in Ba Ria-Vung Tau Province, Vietnam, from May to July 2022.The scientific name of the material was identified as P. adenophylla by Dr. Dang Van Son.A voucher specimen (No UE-P018) was deposited in the herbarium of the Department of Organic Chemistry, Faculty of Chemistry, Ho Chi Minh University of Education, Ho Chi Minh City, Vietnam.

Molecular Docking Studies
The PDB structures of proteins (4j5t and 1t2p) were downloaded from the Protein Data Bank (PDB), while the 3D structures of ligands were modeled via the website chemicalize.com.After the conversion from PDB files into PDBQT format using AutodockTools, the docking study was designated on AutoDock4.2using the Lamarckian genetic algorithm with 250 runs; the maximum number of evals was 25,000,000 (long) for each ligand-protein complex.The configurations with the most repetitions were employed to extract the estimated free energy as a scoring function for predicting the binding affinities to the macromolecular targets.Docking was carried out using the Maestro 12.5 software of Schrodinger Suites (Schrödinger Release 2020-3: Maestro, Schrödinger, LLC, New York, NY, USA, 2020).The protein of Saccharomyces cerevisiae alpha-glucosidase was obtained from the Uniprot database (P53341•MAL12_YEAST) and prepared using the Protein Preparation Wizard protocol.Next, seven reagents and an acarbose reference were generated and prepared using Ligprep [57].As the protein itself does not contain ligands, the grid generation was based on Sitemaps detection's suggestion, incorporating key residues such as Asp 214, Glu 276, and Asp 349 in the binding sites, with the highest site score of 1.1 [58].In addition, the absence of a co-crystallized ligand in a binding site of the protein may not accurately represent the appropriate chemical space for ligand entry.Therefore, rigid docking can potentially lead to misinterpretation of ligand binding modes and inhibitory capacities.In contrast, Induced Fit docking (IFD) [59] allows the binding residues to dynamically adjust their positions to accommodate the ligands, allowing them to penetrate deeper into the binding sites.In the IFD process, specific restraints are implemented to prioritize the formation of h-bonds between the ligands and Asp 214, Glu 276, or Asp 349.After a glide docking SP (standard precision) with IFD, fine-tuning was performed using XP (extra precision)-IFD.The MM-GBSA method [60] was used to calculate the top two poses with the lowest docking scores for each ligand, and the result was chosen based on the best experimental fit.The experimental binding affinity was calculated based on the following equation: IC 50 is the concentration of a drug or inhibitor needed to inhibit a biological process or response by 50%.

Structure Elucidation of the Compounds
Gravity column chromatography was performed on silica gel 60 (0.040-0.063 mm, Merck, Darmstadt, Germany).Thin-layer chromatography (TLC) for checking the chromatographic patterns of fractions and isolated compounds was carried out on silica gel 60 F 254 (Merck, Darmstadt, Germany) and the spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating.Specific rotations were obtained on a Jasco P-1010 polarimeter (Oklahoma City, OK, USA).The high-resolution electrospray mass spectra (HR-ESI-MS) were recorded using a MicrOTOF-Q mass spectrometer (Bruker, MA, USA).The Nuclear Magnetic Resonance (NMR) spectra were measured using a Bruker Avance 500 MHz spectrometer (Bruker, MA, USA).4.7.HPLC Experiments Detected the Presence of Lignins in P. adenophylla High-performance liquid chromatography (HPLC Agilent 1260 Infinity II) using the detector Diode Array detector (DAD) was employed for the analysis.A total of 35 µL of each sample (at the concentration of 1 mg/mL) was injected separately.A gradient system ofIN and water was used during the 60 min analysis: 5% to 10% ACN in 5 min, 10% to 30% ACN in 15 min, 30% to 80% ACN in 10 min, 80% to 100% ACN in 5 min, and 100% A in 5 min.A Luna C18 column (Phenomenex, 150 mm × 4.6 mm.i.d., 5 µm) and a C18 guard column (Phenomenex, Torrance, CA, USA) were employed for this analysis.

Conclusions
Twenty compounds were isolated from D. linearis and P. adenophylla plants using a bioactive-guided isolation procedure.Compounds DL1, DL3-DL10, and PA1-PA6 were reported for the first time in the genera Dicranopteris and Psychotria.The isolated compounds were evaluated for alpha-glucosidase inhibition.Compounds DL3, DL5-DL8, DL10-DL13, and PA4-PA6 showed potent inhibition with IC 50 values ranging from 67.1 to 282.1 µM.A molecular docking study was applied to compounds DL3, DL5, DL8, PA5, and PA6 for elucidating the mechanisms of alpha-glucosidase inhibition.Consequently, this study illustrated that D. linearis and P. adenophylla might be good potential natural sources to develop new antidiabetic preparations.

Table 3 .
The XP-IFD docking scores, the experimental/estimated binding affinities, and the number of H-bonds networks in the ligand-protein complex.